Performance of Immatures of Three Neotropical Miridae at Five Different Temperatures, Reared on Ephestia Kuehniella Eggs on Tobacco Plants

Bulletin of Insectology 71 (1): 77-87, 2018 ISSN 1721-8861

Performance of immatures of three Neotropical Miridae at five different temperatures, reared on Ephestia kuehniella eggs on tobacco plants

1 1 2 1 Vanda Helena Paes BUENO , Flavio Cardoso MONTES , Marcus Vinicius SAMPAIO , Ana Maria CALIXTO , 3 Joop C. VAN LENTEREN 1Laboratory of Biological Control, Department of Entomology, Federal University of Lavras, MG, Brazil 2Institute of Agricultural Sciences, Federal University of Uberlândia, MG, Brazil 3Laboratory of Entomology, Wageningen University, The Netherlands

Abstract

Effects of temperature (16, 20, 24, 28 and 32 ± 1 °C), host plant (Nicotiana tabacum L.) and factitious prey (eggs of Ephestia kuehniella Zeller) on immature development of three recently found Neotropical mirids, Campyloneuropsis infumatus (Carvalho), Engytatus varians (Distant) and Macrolophus basicornis (Stal) were studied at RH 70 ± 10% and 12h photophase in climate cabi- nets. These mirids are being evaluated for biological control of the South American tomato borer Tuta absoluta (Meyrick) and other pests on tomato. Survival of eggs of the three mirid species on tobacco was high (> 80%) at 16-28 °C, but lower (< 80%) at 32 °C. Development times decreased with increasing temperature from 16-28 °C. Nymphal survival was higher (84-96%) at 20, 24 and 28 °C than at 16 and 32 °C (46-83%). The sex ratio of C. infumatus was strongly female biased at all temperatures, whereas it was 1:1 for the other two species. The lower temperature thresholds for egg-adult development of C. infumatus, E. varians and M. basicornis were 9.4, 9.4 and 7.9 °C, and their thermal constants were 384.6, 384.6 and 476.2 DD, respectively. Temperatures between 24 to 28 °C are best for immature performance and for rearing of these mirids species. Eggs of the factitious host E. kuehniella provide adequate food for their mass production. Optimal temperatures for best mirid predator performance are similar to those for the pest T. absoluta, indicating good climate matching.

Key words: Campyloneuropsis infumatus, Engytatus varians, Macrolophus basicornis, Tuta absoluta, biological control, mass production, thermal constants.

Introduction coris tenuis (Reuter) (Hemiptera Miridae) are effec- tively controlling T. absoluta and Bemisia tabaci (Gen- Tomato Solanum lycopersicum L. (Solanaceae) is the nadius) (Calvo et al., 2012). In Brazil, the species Cam- most widely produced fruit vegetable in the world and pyloneuropsis infumatus (Carvalho), Engytatus varians also plays an important role in the agricultural economy (Distant) and Macrolophus basicornis (Stal) (Hemiptera of Brazil, one of the main tomato producers worldwide Miridae) were recently found in the laboratory and field (Agrianual, 2016). Tomato can be attacked by various to prey on eggs and larvae of T. absoluta (Bueno et al., insect pests, including the South American tomato borer 2013a; 2013b; van Lenteren et al., 2017), nymphs of Tuta absoluta (Meyrick) (Lepidoptera Gelechiidae), B. tabaci and eggs and larvae of various other lepidop- which may cause complete crop loss without pest con- teran pests of tomato (Bueno et al. 2013a). Bueno et al. trol (Guedes and Picanço, 2012). In Brazil, T. absoluta (2012; 2013a; 2013b) and Silva et al. (2016) reported is controlled by frequent pesticide sprays (Thomazini et that these Neotropical mirids can use tomato plants as al., 2001) and as a consequence, Brazilian tomato borer an oviposition substrate, can complete their develop- populations have acquired resistance to several active ment on it, and, contrary to other predators, like Geoco- ingredients, resulting in a so-named pesticide treadmill, ris punctipes (Say) (Hemiptera Geocoridae) and Orius decimation of natural enemies and high residue levels insidiosus (Say) (Hemiptera Anthocoridae), can easily on the tomato fruit (van den Bosch, 1978; Siqueira et walk on the stems of tomato plants despite the presence al., 2000; 2001; Silva et al., 2011; Guedes and Picanço, of sticky and poisonous trichomes (Bueno et al., 2013a). 2012). The rapid development of resistance to fre- Predatory Miridae show the characteristic of zoophyto- quently applied pesticides necessitates a search for al- phagy: in addition to eat animal prey, they also feed on ternative control methods, such as biological control. plant tissues to complement or supplement nutritional Heteropteran predators, particularly those belonging to needs or as a source of water (Wheeler, 2001; Albajes the family Miridae (Calvo et al., 2012; van Lenteren, and Alomar, 2008; Bueno and van Lenteren, 2012). 2012; Jaworski et al., 2013; Pazyuk et al., 2014; van Phytophagy can result in both beneficial and detrimental Lenteren et al., 2018a) might provide control of T. abso- effects. Beneficial because plant feeding allows them to luta, while these polyphagous predators may also be survive periods of low pest abundance, detrimental be- used for control of other key pests of tomato (Calvo et cause it may result in plant injury and yield loss. The al., 2009; Martínez et al., 2015; Pérez-Hedo et al, two mirids commercially used for biological control of 2015). In Spain, for example, the Palearctic mirid Mac- important pests in greenhouse tomatoes, M. pygmaeus rolophus pygmaeus (Rambur) and Paleaotropic Nesidio- and N. tenuis, may cause serious plant injury under spe-

cific conditions when prey availability is low and preda- for egg-adult development, and sex ratio of emerged tor density is high. Particularly N. tenuis needs special adults of C. infumatus, E. varians and M. basicornis at attention with regard to population management at high five temperatures (16, 20, 24, 28 and 32 ± 1 °C) with densities in combination with low pest populations, be- E. kuehniella eggs as food source and with tobacco as cause extensive plant feeding by this predator results in host plant. This temperature range was selected based necrotic rings on the stems, shoots, leaf petioles and on the optimum temperatures for tomato production in flower stalks, causing abortion of flowers and young Brazil (Naika et al., 2006), though lower and higher fruits, reduced growth of tomato plants and yield loss temperatures may occasionally occur. Both, growers (Calvo et al., 2009). However, when properly managed, producing tomatoes in the field and in greenhouses try the European mirids are considered important biocontrol to adhere at these optimal temperature schedules. agents in tomato IPM and are used on a large scale in the Meditarranean region (Pérez-Hedo and Urbaneja, 2016). Remarkably, nymphs and adults of the three Materials and methods zoophytophagous Neotropical mirids appeared to cause little injury to tomato seedlings and fruit, even when Rearing of the mirid predators present in high densities and in the absence of prey in Adult mirids (C. infumatus, E. varians and M. basi- the laboratory (Silva et al., 2017a) and in presence of cornis) were collected in areas cultivated with tomato prey in the greenhouse (van Lenteren et al., 2018b). (S. lycopersicum) and tobacco Nicotiana tabacum L. However, the potential of these Neotropical mirids to (Solanaceae) (Bueno et al., 2012). Subsequently, a rear- control pest populations of T. absoluta in the field still ing colony was set up in the laboratory as described in remains to be demonstrated. Knowledge about the Bueno et al. (2013a). Tobacco plants, N. tabacum cv predator’s activity at a range of temperatures occurring TNN were grown in a greenhouse on organic substrate in the field and in greenhouses in tomato production ar- (75% Pinus rusk and 25% vermiculite). Seedlings with eas of Brazil is important to be able to determine their two pairs of leaves were transplanted to 2 L plastic pots potential as natural enemies for control of T. absoluta and maintained until attaining a height of 25 cm. Field and other pests on tomato. Also, information about their collected adults of each predator species were individu- lower temperature threshold and thermal constant assists ally released in acrylic cages (60 × 30 × 30 cm) contain- in determining their periods of activity in the field. ing a tobacco plant as oviposition substrate and a water Hughes et al. (2010) reported that a predator would source. Tobacco plants and the adult predators remained have a selective advantage over its prey if it has a lower in the cages for seven days. Then, the plants containing temperature threshold and a smaller thermal constant mirid eggs were transferred to new cages. Eggs of than its prey, because it would have a greater number of E. kuehniella were offered ad libitum as food to the mirid generations per year and would be active over a wider nymphs and adults twice weekly. The stock rearings range of temperatures than the prey. On the other hand, were kept in a climate room at 25 ± 1 °C, RH 70 ± 10% synchronization of the life history of the predator to that and 12 h photophase. of its prey is important at a range of local tomato production climate conditions (van Lenteren, 2010; Clarke, Development, survival and morphological aspects 2017). Interestingly, Horn (1998) found that biological of eggs control agents often have optimal development tempera- Ten pairs of each predator species were released in tures that are different from their prey. glass pots (1.7 L) containing a plastic cup (200 mL) Mass rearing of natural enemies is a critical step in a (8.5 cm diameter × 5.5 cm height) with a tobacco seed- biological control programme. Thus, information about ling with two pairs of leaves as oviposition substrate responses to different temperatures and thermal re- and E. kuehniella eggs as food ad libitum for a 24h pe- quirements of these Brazilian mirid predators can also riod. The glass pots were sealed with voile fabric and assist in designing an efficient mass rearing system and kept in a climate room at 25 ± 1 °C, RH 70 ± 10% and in estimating the success of establishment after release 12 h photophase. After 24 h, the number of eggs was in tomato crops. Further, we were interested in the qual- counted using a stereomicroscope (40×). Eggs on to- ity of eggs of Ephestia kuehniella Zeller (Lepidoptera bacco leaves, petioles and stems could be seen by put- Pyralidae) as a factitious prey for these predators, be- ting the seedling on a light source, which renders the cause mass rearing of these predators is much easier and eggs darker than the plant tissue, and by looking for cheaper on this factitious prey than, for example, on the opercula which protrude from the plant tissue (Bueno et pest T. absoluta (Mollá et al., 2014). We are aware that al., 2013a). Subsequently, the roots of the seedlings several other factitious prey species, either alone or in were wrapped in moistened cotton in order to avoid combination with artificial media, have been tested to wilting and transferred to Petri dishes (15 cm) sealed rear other mirid species (e.g. Vandekerkhove et al., with PVC film. For each predator species and each tem- 2011; Aubrey at al., 2015), but we have limited our first perature (16, 20, 24, 28 and 32 ± 1 °C), 10 Petri dishes experiments to the easily available, often used and well with a seedling containing eggs were put in climate known flour moth E. kuehniella. chambers with a RH of 70 ± 10% and a 12 h photo- In this paper, data are provided for the development phase. Egg development time and egg survival, ex- time and survival of eggs and nymphs, weight of the 4th pressed by the percentage of 1st instar nymphs hatching, and 5th nymphal instars, egg-adult development time, was determined for each predator species. To be able to lower developmental thresholds and thermal constants study morphological characteristics, eggs of C. infu-

matus, E. varians and M. basicornis were removed from The data for egg and nymphal development and egg to the longitudinal veins of the leaves of tobacco seedlings adult development were adjusted to Poisson distribu- with the aid of stylus and observed using a stereomicro- tions with a logarithmic link function. The other data scope (30×) (Wheeler, 2001). (survival of nymphs and weight of the 4th and 5th instars of E. varians and the 4th instar of M. basicornis) were Development, survival, size of nymphs, sex ratio at adjusted to Quasi-Poisson distributions with a logarith- the adult emergence, and weight of 4th and 5th in- mic link function. The Wald χ2 test was used to test for star nymphs significance of the effects of the models and the pa- To obtain newly-hatched nymphs, potted N. tabacum rameters of the regression analyses. plants 25 cm in height were kept in acrylic cages We analyzed only the effect of temperature on weight (60 × 30 × 30 cm) along with 100 females and 100 of the 4th and 5th instars of E. varians and the 4th instar males of each adult predator species for a period of 24 h of M. basicornis, but we considered temperature effect, in climate chambers at 16, 20, 24, 28 and 32 ± 1 °C, RH species of mirid predator and their interaction as fixed 70 ± 10% and 12 h photophase. Next, plants containing factors for the other analyzed data (egg, nymphal and eggs were transferred to new acrylic cages and were egg to adult development and survival of nymphs). Re- kept at the same conditions. These plants were observed gression analysis was used for evaluation of the tem- daily at the same time for hatched nymphs. One hundred perature effect on egg, nymphal and egg to adult devel- newly-hatched nymphs of each predator species were opment and survival of nymphs. The Dunn test was individually placed in Petri dishes (5 cm diameter) con- used to compare means of egg to adult development taining N. tabacum leaf discs (4.5 cm diameter) on 1% time and survival of nymphs among the three predator agar-water layer and E. kuehniella eggs ad libitum. Each species for each temperature, and to compare weight of Petri dish was sealed with a paper towel secured by a the 4th and 5th instars of E. varians and the 4th instar of rubber band to prevent escape of the nymphs. The leaf M. basicornis among temperatures. discs were changed twice a week, and at the same time Variables that met the assumptions of normality and eggs of E. kuehniella were supplied ad libitum. Each 10 homogeneity were analyzed by ANOVA (survival of nymphs were considered a replicate in a total of 10 rep- eggs, weight of 4th and 5th instars of C. infumatus and licates for data analysis. Instar changes and nymphal weight of 5th instar of M. basicornis). Temperature, spe- mortality were observed daily at the same time using a cies of mirid predator and their interaction were in- stereomicroscope (30×). For each temperature, the de- cluded as independent variables and analyzed by a two- velopment time of each instar, total nymphal develop- way ANOVA for egg survival. Data were transformed ment, egg-adult development, nymphal survival and sex according to y = arcsin √(x / 100) and regression analy- ratio of the adults at emergence were determined. The sis was used to analyze the effect of temperature on egg weight of 20 individuals of the 4th and 5th nymphal survival. A one-way ANOVA and a Tukey test were instars was determined by individually placing nymphs applied to analyze the effect of temperature on the in plastic tubes on an analytical precision scale weight of 4th and 5th instars of C. infumatus and weight (d-0.0001g) (Shimadzu, AW 220). Nymphal instar of 5th instar of M. basicornis. Data of the 4th instar of characteristics as size (measured by the largest width of C. infumatus and the 5th instar of M. basicornis were the cephalic capsule between the outer margins of the transformed according to y=√x. To determine deviations compound eyes using a micrometer reticule coupled in from a 1:1 male/female sex ratio, the data were analyzed stereomicroscope, 40×) and body colour (observed by by a homogeneity χ2 test. For all analyses, a P = 0.05 stereomicroscope at 30×) were recorded. was used as significance level. The lower temperature thresholds for development Data analysis (LDT) and the thermal constants (K) of the three mirid Data for cohorts of 100 nymphs (10 nymphs per repli- species were calculated with the hyperbole method. This cate) were used to determine the development time of method makes use of a linear regression y=a+bx, where each instar, total nymphal development time, nymphal y is the reciprocal of the development time in days and x survival and sex ratio of the adults at emergence for is the temperature in degrees Celsius (Campbell et al., each predator species at each temperature. All statistical 1974; Bergant and Trdan, 2006). To estimate the lower procedures were performed using the software R 3.2.0 temperature threshold with this method, measurements (R Development Core Team, 2015). First, the data were are needed for at least four different temperatures in the subjected to an exploratory analysis to verify the resid- range of insect development (Campbell et al., 1974), ual normality with the Shapiro-Wilk test, and then the and as we have data for five temperatures, we have met data were tested for residual homogeneity with the this prerequisite. Levene test (Gujarati, 2004); both tests were performed at P = 0.01 and P = 0.05. Variables with residuals not normally distributed and/or heteroscedastics (develop- Results ment of eggs, total development of nymphs and egg to adult development, survival of nymphs and weight of Morphology, development time and survival of eggs the 4th and 5th instars of E. varians and the 4th instar of Females of C. infumatus, E. varians and M. basicornis M. basicornis) were analyzed with Generalized Linear oviposit in the main midrib of the leaves, and rarely in Models (Nelder and Wedderburn, 1972; McCullagh and the petioles and stems of tobacco plants. The eggs are Nelder, 1989; Lee et al., 2006). inserted into the plant tissue with only their opercula

visible. Eggs of C. infumatus are elongated and slightly velopment time decreases with increasing temperature curved, 0.762 mm long and 0.254 mm wide, milky- (figure 1). Development times of the eggs of the three white to translucent with a smooth outer egg surface and mirids did not significantly differ at each test tempera- two short respiratory extensions (spiracles), one on the ture (P = 0.0549, df = 2; 135; table 1). Also, no signifi- concave region and another on the convex region near cant interaction was found between temperature effect the operculum. Eggs of E. varians are 0.915 mm long and species of mirid for egg survival (F8;135 = 0.0840, and 0.254 wide, are milky-white to pale yellow and P = 0.9438). Further, no significant differences were have only one spiracle in the concave region near the found for egg survival (F2;135 = 0.351, P = 0.9185) of the operculum, which is longer than those of C. infumatus three mirid species at each temperature. But temperature eggs. Eggs of M. basicornis are similar to those of significantly affected egg survival (F4;135 = 15.763, E. varians, 0.813 mm long and 0.254 mm wide, and P < 0.0001) of the mirids, and egg survival was highest milky-white to translucent in colour. at the intermediate temperatures (figure 2). No significant interaction was found between tem- Because no statistical differences existed between spe- perature effect and species of mirid for the development cies at a certain temperature in egg development time, time of eggs (P = 0.5457, df = 8; 135). However, tem- egg survival and development time of nymphs (table 1), perature significantly affected the development time of we have combined the data of the three species to calcu- eggs (P < 0.0001, df = 4; 135) of the three mirids: de- late the relationships represented in figures 1, 2 and 3.

Figure 1. Developmental time of eggs in days (means ± SE) of C. infumatus, E. varians and M. basicornis at five temperatures, RH 70 ± 10% and 12h photophase. f(x)=exp(5.2862259-0.1792171x+0.0022983x2).

Table 1. Developmental time in days (means ± SE) of eggs, nymphal instars, total nymphal development (TND) and egg-adult development of C. infumatus, E. varians and M. basicornis at five temperatures, RH 70 ± 10% and 12h photophase; n = number of individuals.

Nymphal instars* T °C Species Eggs* n TND* Egg-adult** n 1st n 2nd n 3rd n 4th n 5th n C. infumatus 18.5±1.37 400 6.3±0.13 95 7.2±0.17 89 7.6±0.21 81 8.3±0.10 78 12.8±0.19 64 41.7±0.92 60.2±0.77A 64 16 E. varians 19.3±0.91 498 6.5±0.13 95 6.4±0.15 81 7.4±0.19 73 9.1±0.18 68 13.3±0.31 51 42.0±1.15 61.3±1.21A 51 M. basicornis 22.6±1.10 258 7.3±0.11 97 6.5±0.14 92 7.1±0.16 87 8.0±0.16 74 12.2±0.24 71 39.0±2.04 61.6±2.22A 71 C. infumatus 13.0±1.16 190 4.0±0.72 100 3.3±0.05 99 3.4±0.06 98 3.7±0.08 97 6.3±0.05 93 20.3±0.15 33,3±0.31B 93 20 E. varians 13.6±1.01 218 4.4±0.56 98 3.3±0.05 97 3.4±0.06 91 3.8±0.04 91 6.0±0.03 89 20.9±0.18 34.5±0.40B 89 M. basicornis 14.6±0.91 213 5.5±0.67 100 3.7±0.05 100 3.8±0.04 100 4.3±0.05 97 7.2±0.05 92 24.7±0.33 39.3±0.42A 92 C. infumatus 9.9±0.42 250 3.2±0.04 100 2.3±0.05 100 2.5±0.05 97 3.0±0.07 97 4.4±0.06 96 16.1±0.23 26.0±0.29B 96 24 E. varians 10.0±0.40 132 3.6±0.05 99 2.5±0.07 96 2.5±0.05 93 3.2±0.06 90 4.7±0.05 84 17.2±0.59 27.2±0.63B 84 M. basicornis 10.7±0.21 322 3.2±0.04 100 3.3±0.05 97 3.3±0.06 96 3.5±0.07 94 5.2±0.04 93 18.2±0.20 28.9±0.20A 93 C. infumatus 7.5±0.36 237 3.1±0.05 100 2.2±0.06 100 1.9±0.05 100 2.2±0.05 96 3.7±0.07 92 12.9±0.23 20.4±0.26B 92 28 E. varians 7.1±0.35 216 2.9±0.03 100 2.0±0.03 100 1.5±0.05 98 2.4±0.05 97 3.2±0.05 94 12.6±0.27 19.7±0.23B 94 M. basicornis 8.2±0.71 221 3.1±0.03 99 2.2±0.05 99 2.3±0.05 98 2.5±0.05 96 4.1±0.05 94 15.0±0.21 23.2±0.24A 94 C. infumatus 7.0±0.51 185 3.3±0.06 94 1.9±0.05 89 1.9±0.04 83 2.6±0.07 74 3.7±0.09 45 13.3±0.37 20.4±0.36A 45 32 E. varians 6.1±0.14 352 2.1±0.03 100 1.8±0.03 99 1.9±0.02 92 2.0±0.04 87 3.2±0.07 51 11.2±0.20 17.3±0.22B 51 M. basicornis 7.2±0.21 146 2.8±0.03 98 1.9±0.02 96 2.0±0.02 92 2.3±0.05 91 3.8±0.05 83 13.1±0.18 20.3±0.20A 83

*Means were not significantly different when comparing the species at the same temperature (Wald χ2 test; p > 0.05). **Means followed by the same capital letters in columns are not significantly different when comparing the species at the same temperature (Dunn test; p > 0.05).

Figure 2. Fraction survival of eggs (means ± SE) of C. infumatus, E. varians and M. basicornis at five temperatures, RH 70 ± 10% and 12h photophase. y= -0.2134x2+9.5603x–15.045, R² = 0.9403.

Figure 3. Developmental time of nymphs in days (means ± SE) of C. infumatus, E. varians and M. basicornis at five temperatures, RH 70 ± 10% and 12h photophase. f(x)=exp(7.5439642–0.3249259x+0.0052779x2).

Development time of instars, total nymphal devel- 135). Egg-adult development of the three mirid species opment time, egg-adult development time, nym- was not different at 16 °C (Dunn = 1.0245, df = 2, P = phal survival and thermal requirements 0.6000), but development of M. basicornis was longer No significant interaction was found between tempera- than that of E. varians at the other four temperatures, ture effect and species of mirid for total nymphal devel- and longer than that of C. infumatus at 20 °C (Dunn = opment time (P = 0.1733, df = 8; 135). Also, no signifi- 21.1718, df = 2, P < 0.0001), 24 °C (Dunn = 14.8803, df cant differences were found for the development time of = 2, P < 0.0001) and 28 °C (Dunn = 20.5157, df = 2, P < instars (P = 0.3358, df = 2; 135) of the three mirid spe- 0.0001) (table 1). The egg-adult development of C. in- cies at each temperature. Temperature affected total fumatus was significantly longer than that of E. varians nymphal development time (P < 0.0001, df = 4; 135), only at 32 °C (Dunn = 18.9966, df = 2, P < 0.0001) (ta- which decreased with increasing temperature from 16 to ble 1). As expected, temperature affected the egg-adult 28 °C and stabilized between 28 and 32 °C (figure 3). In development (P < 0.0001, df = 4; 135) of the three addition, no significant interaction was found between mirids, showing shorter development with increasing temperature effect and species of mirid for egg-adult temperature (figure 4). development (P = 0.2785, df = 8; 135). Egg-adult de- A significant interaction was found between tempera- velopment was significantly different among the three ture effect and species of mirid for survival of the mirid species at some temperatures (P = 0.0321, df = 2; nymphs (P = 0.0049, df = 8; 135). The percentage nym-

Figure 4. Developmental time egg-adult in days (means ± SE) of three species of Miridae at five temperatures, RH 70 ± 10% and 12h photophase. C. infumatus f(x)=exp(7.3645425–0.2729148x+0.0042136x2), E. varians f(x)=exp(7.3645425−0.0010038–0.2729148x+0.0042136x2); M. basicornis f(x)=exp(7.3645425+0.0779611– 0.2729148x+0.0042136x2). The dots represent egg-adult development time of the pest T. absoluta (data from Barri- entos et al.,1998).

Table 2. Percentage survival of eggs and nymphs (means ± SE) and estimated total survival (%) of immatures of C. infumatus, E. varians and M. basicornis at five temperatures, RH 70 ± 10% and 12h photophase; n = number of individuals.

Survival (%) Estimated total T °C Species Eggs* n Nymphs** n survival of immatures*** C. infumatus 84.2 ± 1.81 400 64.0 ± 3.71AB 64 53.8 16 E. varians 83.5 ± 1.97 498 51.0 ± 4.06B 51 42.5 M. basicornis 82.3 ± 2.58 258 71.0 ± 4.33A 71 58.4 C. infumatus 87.3 ± 2.53 190 93.0 ± 2.60A 93 81.1 20 E. varians 90.2 ± 2.34 218 89.0 ± 1.79A 89 80.2 M. basicornis 91.7 ± 2.05 213 92.0 ± 2.90A 92 84.3 C. infumatus 95.7 ± 1.07 250 96.0 ± 2.21A 96 91.8 24 E. varians 92.3 ± 1.73 132 84.0 ± 4.26B 84 77.5 M. basicornis 94.3 ± 1.02 322 93.0 ± 2.13AB 93 87.4 C. infumatus 84.2 ± 4.54 237 92.0 ± 2.49A 92 77.4 28 E. varians 82.5 ± 4.66 216 94.0 ± 2.21A 94 77.5 M. basicornis 81.8 ± 3.38 221 94.0 ± 2.21A 94 76.8 C. infumatus 71.8 ± 6.62 185 46.0 ± 6.00B 46 33.0 32 E. varians 76.4 ± 4.92 352 51.0 ± 4.33B 51 38.9 M. basicornis 71.2 ± 5.10 146 83.0 ± 3.35A 83 59.0

*Means were not significantly different when comparing the three species at the same temperature (F test; p > 0.05). **Means followed by the same capital letters in columns are not significantly different when comparing species at the same temperature (Dunn test; p > 0.05). ***% estimated total immature survival (% eggs × % nymphs/100).

phal survival of C. infumatus, E. varians and M. basi- = 8.2576, df = 2, p = 0,020) than C. infumatus and cornis (P < 0.001, df = 2; 135) was high (84-96%) and M. basicornis at 16 °C (table 2, figure 5). The estimated similar at 20 °C (Dunn = 2.3192, df = 2, p = 0.311), total percentage survival of immatures of the three 24 °C (Dunn = 5.7068, df = 2, p = 0.060) and 28 °C mirids was lowest at 32 °C (33-60%) and 16 °C (Dunn = 0.4554, df = 2, p = 0.800) (table 2). C. infu- (42-58%) and highest at 20, 24 and 28 °C (77-87%) (ta- matus and E. varians showed lower survival than ble 2). M. basicornis at 32 °C (Dunn = 17.6813, df = 2, The development rate of three mirid species as a p < 0.001), and E. varians showed lower survival (Dunn function of temperature is presented in table 3. The

Figure 5. Fraction survival of nymphs (means ± SE) of three species of Miridae at five temperatures, RH 70 ± 10% and 12h photophase. C. infumatus f(x)=exp(−17.552295+1.735792x−0.037080x2)/[1+exp(−17.552295+1.73579 2x- 0.037080x22)]; E. varians f(x)=exp(−17.552295−1.488780+1.73579 2x+0.049655x−0.037080x2)/[1+exp (−17.552295−1.488780+1.735792x+0.049688x−0.037080x2)]; M. basicornis f(x)=exp(−17.552295−1.333229+ 1.735792x0.083811x−0.037080x2)/[1+exp(−17.552295−1.333229+1.735792x+0.083811x−0.037080x2).

Table 3. Regression equations and their correlation coefficients (R2) used to calculate the lower developmental temperature thresholds (LTD) and thermal constants (K), for the different developmental stages of C. infumatus, E. varians and M. basicornis.

Species Stage LTD (°C) K(DD) Regression equation R2(%) Egg 7.94 156.25 y=0.0064x-0.00508 0.99 C. infumatus Nymph 9.81 232.56 y=0.0043x-0.0422 0.98 Egg-adult 9.35 384.62 y=0.0026x-0.0243 0.99 Egg 9.31 138.89 y=0.0072x-0.0671 0.99 E. varians Nymph 9.21 243.90 y=0.0041x-0.0378 0.98 Egg-adult 9.42 384.62 y=0.0026x-0.0245 0.99 Egg 8.65 166.67 y=0.006x-0.0519 0.99 M. basicornis Nymph 7.40 321.50 y=0.0032x-0.0237 0.99 Egg-adult 7.88 476.19 y=0.0021x-0.0165 0.99

values of the lower developmental temperatures (LTD) were slightly bigger (0.300, 0.376, 0.458 and 0.525 mm). of the development stages were different for all three Differences in weight were found for the 5th nymphal mirid species (table 3). The values of LTD were lowest instars of each mirid species reared at the five tempera- for the species M. basicornis. The thermal constants tures, but no clear trends were discovered, except that (K) for egg-adult development of C. infumatus and weight was generally lowest at the highest temperature E. varians were similar and lower than that of M. basi- (table 4). C. infumatus showed the lowest weights for th th cornis (table 3). The thermal constant for the nymphal the 4 (F4;95 = 32.2310, P < 0.0001) and 5 nymphal in- stage of M. basicornis was higher than that of C. infu- stars (F4;95 = 3.8040, P < 0.0065), at 32 °C. E. varians matus and E. varians (table 3). showed the lowest weight of the 4th (Dunn = 35.4221, df = 4, P < 0.0001) and 5th nymphal instars at 16 °C (Dunn Nymphal instar characteristics and weight of 4th = 63.1319, df = 4, P < 0.0001). M. basicornis showed and 5th nymphs lowest weight of the 4th (Dunn = 63.7721, df = 4, st th Newly emerged 1 instar nymphs of C. infumatus and P < 0.0001) and 5 (F4;95 = 43.5140, P < 0.0001) nym- M. basicornis were light green, whereas nymphs of phal instars at 16 °C and 32 °C (table 4). E. varians were yellow. Nymphs of all three mirid species have brown-red eyes. First instar nymphs are of Sex ratio similar size (0.221 mm, measured by the largest width of Temperature did not significantly affect sex ratios of the cephalic capsule between the outer margins of the the three mirid species. E. varians and M. basicornis compound eyes). The 2nd, 3rd, 4th and 5th nymphal instars exhibited a sex ratio which did not significantly differ of C. infumatus and M. basicornis were similar in ce- from 1:1 at the five temperatures, whereas the sex ratio phalic capsule size (0.284, 0.292, 0.420 and 0.435 mm, of C. infumatus was significantly female biased at all respectively), while the respective instars of E. varians temperatures (table 5).

Table 4. Weight (mg) of 4th and 5th nymphal instars (means ± SE) of C. infumatus, E. varians and M. basicornis at five temperatures, RH 70 ± 10% and 12h photophase, n = 20 individuals.

Weight of nymphal instar (mg) T °C Species 4th 5th 16 0.970 ± 0.012A 1.375 ± 0.031A 20 0.810 ± 0.026B 1.210 ± 0.041B 24 C. infumatus 0.815 ± 0.024B 1.285 ± 0.024AB 28 0.700 ± 0.251C 1.300 ± 0.034AB 32 0.610 ± 0.028C 1.240 ± 0.027B 16 0.870 ± 0.026c 1.370 ± 0.029d 20 0.845 ± 0.023c 1.715 ± 0.033bc 24 E. varians 1.000 ± 0.029ab 1.970 ± 0.032a 28 0.895 ± 0.026bc 1.885 ± 0.043ab 32 1.055 ± 0.019a 1.620 ± 0.042c 16 0.750 ± 0.019b 1.075 ± 0.032C 20 0.920 ± 0.015a 1.380 ± 0.036B 24 M. basicornis 0.810 ± 0.216b 1.580 ± 0.031A 28 0.775 ± 0.012b 1.185 ± 0.024C 32 0.565 ± 0.024c 1.130 ± 0.031C

Means followed by the same capital (Tukey test) or small (Dunn test) letters in columns are not significantly different when comparing each species among temperatures (p < 0.05). The Tukey test was applied when the data showed a normal distribution, the Dunn test was applied on data with a non-normal distribution.

Table 5. Sex ratio expressed as a proportion of females cornis nymphs showed a high survival at all tempera- [SR = ♀ / (♀ + ♂)] of C. infumatus, E. varians and tures, but, like the other two mirids, was lighter at the M. basicornis at five temperatures, RH 70 ± 10% and lowest and highest temperature. E. varians appeared 12h photophase. least adapted to the lowest temperature, indicated by low survival and weight. Only sex ratio was not affected T °C Species Sex ratio χ² by temperature. Temperature extremes of 16 °C and 16 0.67 (43/21) 7.5625* 32 °C had in most cases a negative effect on develop- 20 0.87 (81/12) 51.1936** ment and survival of eggs and nymphs. The negative 24 C. infumatus 0.86 (83/13) 51.0417** effects at the lowest temperature are in agreement with 28 0.84 (78/14) 44.5217** findings of Roy et al. (2002), who concluded that expo- 32 0.73 (34/12) 10.5217** ns sure of insects to low temperature affects all of their de- 16 0.63 (33/19) 3.7692 velopment stages, with a reduction in the metabolic rate 20 0.41 (37/52) 2.5281ns ns and, consequently, with slower development. However, 24 E. varians 0.55 (47/37) 1.1905 all three species were still developing and showed im- 28 0.50 (47/47) 0.0000ns ns mature survival at the lowest (> 42.5%) and highest 32 0.52 (27/24) 0.1765 (> 33%) temperatures, so they remain active at the op- 16 0.54 (39/32) 0.6901ns ns timal Brazilian temperature regime for tomato produc- 20 0.47 (44/48) 0.1739 tion (Naika et al., 2006). 24 M. basicornis 0.59 (55/38) 3.1075ns ns Though we also found negative effects at the highest 28 0.52 (49/45) 0.1702 temperature, egg survival values at 32 °C for C. infu- 32 0.56 (47/36) 1.4578ns matus, E. varians and M. basicornis were still higher Significant difference by χ2 test at *5% (χ2 tabulated = than those found for M. pygmaeus at 30 °C (Perdikis 3.841, df = 1) and **1% (χ2 tabulated = 5.9915, and Lykouressis, 2002; 2004). Host plant effects - we df = 1); ns = not significant by χ2 test. used tobacco, while Perdikis and Lykouressis (2004) used tomato and eggplant plants as oviposition substrates - may partly explain the difference in egg sur- Discussion vival. Another difference between their and our experi- ment is that we offered prey to the egg laying adults, The performance of organisms depends greatly - but not while Perdikis and Lykouressis (2004) did not. Many only - on temperature, with performance generally being species of mirids show endophytic oviposition, and their maximized within a rather narrow thermal range (Angil- eggs, which are embedded in the leaf, are particularly letta, 2009). Temperature affected most variables during vulnerable to desiccation (Wheeler, 2001). Cocco et al. immature development of the three mirid species, and (2008) mentioned that tobacco has a high cell turgidity temperature effects are also reflected in their lower and Constant et al. (1996) observed egg-hatching per- threshold temperatures and thermal constants. C. infu- centages of 81.6% on tobacco, while they found only matus and E. varians nymphs were heavier, and showed 43.4% egg-hatching on geranium, Pelargonium pel- higher survival at intermediate temperatures. M. basi- tatum L. (Geraniaceae), as oviposition substrates for

M. pygmaeus. Our results in combination with the above Copland (1996) reported that diploid species, such as mentioned literature data suggest that tobacco provides hemipterans, show 1:1 sex ratios with little variation. a good oviposition substrate for mirids, whereas tomato, This is what we found for E. varians and M. basicornis. sweet pepper and gerbera might be less suitable. Similar 1:1 ratios were found for the mirids M. pyg- The three mirid species showed five instar stages in- maeus (Perdikis and Lykouressis, 2004), N. tenuis (San- dependent of the temperature to which they were ex- chez et al., 2009) and Tupiocoris cucurbitaceus posed. Previous studies with other mirids, M. pygmaeus (Spinola) (López et al., 2012). However, the sex ratio of (Perdikis and Lykouressis, 2002) and N. tenuis (Ur- C. infumatus was strongly female biased, and as yet, we baneja et al., 2005), also showed five instar stages when have no explanation for this finding. exposed to different temperatures. Exposure of the three In order to be efficient, natural enemies should be able mirids to 16 °C led to increased nymphal and egg-adult to disperse, develop, reproduce and attack the pest under development time, probably due to a slower metabolism the weather conditions in which they are to be used (van at low temperatures. Sinclair et al. (2003) reported that Lenteren, 2010). The pest T. absoluta is active within a metabolic activity as well as the amount of food intake temperature range of 19.7 °C to 27.1 °C, with the short- of insects decreases at low temperatures, while energy est development time at 27.1 °C (23.8 days) (Barrientos expenditure increases. The development times observed et al., 1998). It shows a high egg, larval and pupal sur- for C. infumatus and M. basicornis at 32 °C were simi- vival, and the shortest larval development time at 25 °C. lar to values reported for the mirid Pilophorus typicus Its lower temperature threshold is 8.14 °C and its ther- (Distant) at 30 °C (Nishikawa et al., 2010). Temperature mal constant is 453.60 DD (Barrientos et al., 1998). All effects on nymphal survival were similar to those on these values are in the range of those that we found for development: survival was highest at intermediate tem- the three mirid predators. Thus, the pest optimally de- peratures, and was lower at the extremes, though sur- velops within the same temperature range as the imma- vival of M. basicornis was less affected than that of the ture stages of the predatory mirids. The overlap in per- other two species at the extremes. Sanchez et al. (2009) formance at a range of temperatures is illustrated in fig- reported highest survival for N. tenuis at 25 °C and low- ure 4, where development times of the pest and the est at 15 °C and 35 °C, while no nymphs survived at 40 mirids are given. Climate matching between pest and °C. Nishikawa et al. (2010) observed lower survival predators is a positive finding, as the predators can es- rates of P. typicus at lower (17.5 °C) and higher (30 °C) tablish themselves and develop in crops where the prey temperatures. is present and active. Wheeler (2001) stated that latitudinal and altitudinal Climate matching of pest and predator, together with a effects on the development of mirids species are evi- high predation capacity of the nymphs and adults of the dent. The lower temperature thresholds and thermal three mirids (Bueno et al., 2013a; van Lenteren et al., constants we found for three mirid species were quite 2016) may make these mirids potential candidates for different, despite the fact that they were collected at the biological control of T. absoluta. Based on climate same location, and were exposed to the same food and matching data alone, M. basicornis might be the best rearing conditions. Nevertheless, these three mirid spe- candidate for biological control of this pest, as it has the cies have a wide geographical distribution over the lowest temperature threshold and is performing best at America’s, and may, thus, have regional populations intermediate and extreme temperatures, but we do real- with different temperature thresholds and thermal con- ize, of course, that a number of other predator character- stants like we found. M. basicornis starts nymphal de- istics will determine its eventual success in biocontrol of velopment at a lower temperature threshold and needs T. absoluta. We have recently published on population more degree days than C. infumatus and E. varians. The development of the three mirids on tomato with T. abso- values of the lower temperature thresholds we found luta as prey (Silva et al., 2016), with E. kuehniella as were lower than those recorded for the mirid N. tenuis prey (Bueno et al., 2018), on predation of prey by all (Sanchez et al., 2009; Hughes et al., 2010; Pazyuk et nymphal stages and during the whole adult stage (van al., 2014), indicating that the Neotropical species we Lenteren et al., 2017; 2018c), about the effect of plant studied start performing at lower temperature conditions and fruit feeding by these mirids (Silva et al., 2017a, than the mirids successfully used in biocontrol in van Lenteren et al., 2018b), on the role of chemical Europe. communication in the mirid-pest-plant system (Silva et Although significant differences in weight were found al., 2017b; 2018), as well as on the capacity to reduce for each mirid species reared at different temperatures, pest populations in an experimental greenhouse setting no clear trends were found, with the exception of the (van Lenteren et al., 2018b). All current information of highest temperature where weight was generally lower these three Neotropical mirids is now under assessment than at the other temperatures. This is in agreement with and will be used for selection of the most promising Wheeler (2001), who reported that, in general, more predator for control of T. absoluta and other pests on rapid development at higher temperatures is associated tomato in Brazil. with reduced weight and survivorship. An additional conclusion of this study is that tobacco Temperature did not affect the sex ratio of the three is a good host plant and E. kuehniella eggs are suitable mirids. Lauge (1985) stated that unfavourable condi- factitious prey for the three mirids. Mass rearing of the tions for nymphal development, such as extreme tem- mirids under laboratory conditions is much easier on peratures might affect sex ratios, resulting in fewer fe- tobacco than tomato, and E. kuehniella eggs are easier males, but we did not observe such an effect. Jervis and to obtain than T. absoluta eggs.

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W., 2018c.- Adult lifetime predation of Tuta BioControl, 61: 545-553. absoluta eggs by three Neotropical mirid predators on to- SILVA D. B., BUENO V. H. P., CALVO F. J, VAN LENTEREN J. C., mato.- Bulletin of Insectology, 71 (2): in press. 2017a.- Do nymphs and adults of three Neotropical zoophy- WHEELER A. G., 2001.- Biology of the plant bugs (Hemiptera: tophagous mirid damage leaves and fruits of tomato?- Bulle- Miridae): pests, predators, opportunists.- Cornell University tin of Entomological Research, 107: 200-207. Press, Ithaca and London. SILVA D. B., WELDEGERGIS B., VAN LOON J. J. A., BUENO V. H. P., 2017b.- Comparative analysis of herbivore-induced plant volatiles from tomato plants infested by either Tuta Authors’ addresses: Vanda Helena Paes BUENO (corre- absoluta or Bemisia tabaci. - Journal of Chemical Ecology sponding author: [email protected]), Flavio Cardoso 43: 53-65. MONTES, Ana Maria CALIXTO, Laboratory of Biological Con- SILVA D. B., BUENO V. H. P., VAN LOON J. J. A., PEÑAFLOR M. trol, Department of Entomology, Federal University of Lavras, F. G. V., BENTO J. M. S., VAN LENTEREN J. C., 2018.- Attrac- 37200-000 Lavras, MG, Brazil; Marcus Vinicius SAMPAIO, tion of three mirid predators to tomato infested by both the Institute of Agricultural Sciences, Federal University of tomato leaf mining moth Tuta absoluta and the whitefly Uberlândia, 38408-100 Uberlandia, MG, Brazil; Joop C. VAN Bemisia tabaci.- Journal of Chemical Ecology, 44: 29-39. LENTEREN, Laboratory of Entomology, Wageningen Univer- SINCLAIR B. J., VERNON P., KLOK C. J., CHOWN S., 2003.- In- sity, Wageningen 6708PB, The Netherlands. sects at low temperatures: an ecological perspective.- Trends in Ecology & Evolution, 18: 257-262. Received October 7, 2017. Accepted February 13, 2018.